Establishment Of Analytical Method And Application For Simultaneous Determination Of Four Fat-soluble Vitamins In Human Serum Based On LC-MS/MS

Objective:Vitamins are a class of trace low-molecular organic compounds necessary to maintain the normal function of the body.It cannot be synthesized or synthesized in a small amount in the body and must be supplied by food.It plays an important role in regulating material metabolism.Vitamins are essential substances for maintaining human health.They belong to human endogenous metabolites.Their deficiency or excess will affect the body’s metabolic balance.By detecting the content of vitamins,the nutritional status of the patient’s body can be assessed.The current measurement methods mainly include enzyme immunoassay,fluorescence analysis,liquid chromatography and mass spectrometry.When the liquid chromatography-mass spectrometry technology is used for the actual analysis of endogenous metabolites,the test substance contained in the authentic matrix will affect the concentration of the standard sample,thereby affecting the accuracy of the quantitative results.At present,pretreatment methods such as activated carbon adsorption are often used to obtain a stripped matrix from which endogenous metabolites have been removed from the real biological matrix.However,this method is costly,and may destroy the matrix components other than the endogenous metabolites or the endogenous metabolites are not completely removed,which affects the original information of the sample,which leads to the problem of matrix effect.In addition,some newly developed methods cannot find a suitable blank matrix,which increases the difficulty of quantitative analysis.This study aims to use LC-MS/MS to simultaneously detect the four fat-soluble vitamins of vitamin A,vitamin D2,vitamin D3 and vitamin E in human serum,and establish a rapid and accurate analysis for human endogenous metabolites.An efficient and accurate detection method that meets the clinical needs of large-scale sample detection.Research method: Chromatographic conditions: Column: Poroshell 120 EC-C18(4.6*50mm)2.7-Micron,mobile phase: A liquid: 0.5% formic acid water + 0.5mol/L ammonium formate + water(1:1:8),Liquid B: pure methanol;flow rate: 0.65ml/min,gradient elution: 0.0-0.5min: mobile phase A: B=20:80,0.5-2.1min: mobile phase A:B=20:80,2.1-4.6 min mobile phase A:B=4:96;4.6-6.8min: mobile phase A:B=0:100;6.9-8.9min: mobile phase A:B=20:80,column temperature: 40 ℃,injection volume :20u L.Mass spectrometry conditions: Electrospray ion source(ESI),multiple reaction monitoring(MRM)scan mode,curtain gas(CUR)pressure 103 k Pa,ionization voltage(IS)5500 V,temperature(TEM)set to 350 ℃,spray gas(The pressure of GS1)is 403 k Pa,and the pressure of auxiliary heating gas(GS2)is 445 k Pa.The pretreatment adopts liquid-liquid extraction method to extract the fat-soluble vitamins from the serum,and then carries out the methodological verification.Results: This study established a method that can be used to detect 4 fat-soluble vitamins in human serum at the same time.Calculated with quality control samples,the intra-day precision obtained does not exceed 10%,and the inter-day precision does not exceed 9%.The results show that this method has high precision and accuracy,and the retention time is short.The stability results showed that the RSD did not exceed 9%when the serum was placed at room temperature for 2 hours,3 freeze-thaw cycles,frozen at-20°C for 15 days,and placed at room temperature for 24 hours after treatment,indicating that the sample is in this research method.Down detection is stable.Conclusion: This study took the detection of 4 fat-soluble vitamins in human serum as an example,and explored a new method for analyzing human endogenous metabolites,using bovine serum albumin(BSA)as a blank matrix to add standard and isotope internal standards The method of deducting the background from the endogenous substance by the substance can realize the matrix matching of the standard curve sample and the actual sample to be tested,reducing the interference of matrix effect and making it more convenient.Experiments show that this method meets the methodological evaluation criteria,and has good accuracy and reproducibility.Therefore,for some endogenous compounds,a pure blank matrix cannot be obtained,and the matrix effect problem caused by the mismatch between the standard substance tested under the simulated matrix and the matrix of the sample to be tested,the direct method can be used for detection.